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#MicroTwJC: TCA Cycle and Voltammetry

This weeks Microbiology Twitter Journal club focuses on the electrifying bacterium known as Shewanella. This species of bacteria has been found in iron and heavy metal rich sediments, and can respire metal as we would respire oxygen. This paper is all about respiration, and looks at one part of how organisms convert chemicals into energy that they can use. For a rough overview, check this out

For organisms like us, who use oxygen as the main electron acceptor, we can simply transport the oxygen to our mitochondria where respiration is occurring. But for bacteria like Shewanella, that use iron as their electron acceptors, they can't transfer iron into their intracellular compartments as easily, so they have moved a significant part of their electron transport chain to their surfaces. So when they metabolise compounds, they charge their surface. The bacterium's ability to reduce metals have lead to some exploring how it can be used to detoxify heavy metals in the environment, and others exploring how it can be used in a microbial fuel cell.

But that is not what this research is using Shewanella for. In this research, the bacterium is being used to answer the question of what role electrical signalling plays in respiration.

For instance, the TCA cycle and the electron transport chain are both reliant on having oxygen (Or some other good electron acceptor in the case of Shewanella) being present. So the master regulator genes that determine whether a bacteria should activate its TCA cycle must be certain that the bacterium is in an environment with the right level of electron acceptors present before they cause the TCA cycle to occur.

In order to test whether they are in oxidative conditions, bacteria have the ArcB sensor protein, which can detect the presence of reduced quinolone molecules carrying an extra electron with them. When the levels of these molecules decrease, it is usually because the bacterium is in an oxidative environment in which there are molecules that are ready to accept electrons, and this is when the ArcB sensor activates and tells the cell it can run the TCA cycle.

Whilst all bacteria can regulate their metabolism by sensing the oxidative properties in their environment, it's very difficult to measure how they do it in real time, because most bacteria compartmentalise their electron transport chain systems. Not so with Shewanella, which is why the researchers chose to use it for their experiments.

Voltammetry

The primary focus here is on examining the electrical properties of these bacteria, hopefully so that future researchers can do something interesting with it.

They used a technique known as "Voltammetry" to investigate these properties. In its most basic form, it would involve putting bacteria on an electrode, and trying to measure how much current flows through them if we put different voltages across them.

To do this, the researchers used a three electrode system. The three electrodes each do different jobs.

Working Electrode: This electrode is where the bacteria sit in this system. It applies a voltage to the bacteria to facilitate the transfer of charge through them.

Counter Electode: This needs to balance the voltage from the working electrode in order to allow current to properly flow. It needs to be in balance with the working electrode, and is needed to get a measurement on the current flowing through a system.

Reference Electrode: This electrode has a known electrical potential. The voltage potentials between this electrode and the working electrode are compared in order to get a standardised value of Voltage, which is measured as Voltage vs the voltage of a Standardised Hydrogen Electrode (SHE).

In the first set of experiments shown in the paper, they applied a range of different voltages to these bacteria whilst at the same time measuring the current flowing through the system.

Figure 2 A. Here they measured how voltage varies with current at 1 hour, 5 hour and 20 hour post inoculation. The peak current was at 0.0V at 20 hours post inoculation. At the higher voltages, the flow of current was lower.

Figure 2 B. The authors then decided to ask what would happen if they shut down the TCA cycle. So they added malonic acid, a chemical that blocks one of the key proteins in the TCA cycle (Succinate Dehydrogenase). When they added this, they found that the peak at 0V disappears, suggesting that this peak is due to the TCA cycle.

Figure 2 C. The authors then tried to measure out the current flowing through Shewanella when it had a key protein in the TCA cycle (Succinate Dehydrogenase) deleted. This also didn't have a peak current at 0V.

These suggest that the TCA cycle is most active when the potential is at 0V relative to a Standard hydrogen electrode, and that this is primarily responsible for the flow of current from this bacteria. The researchers separate out the voltage conditions into Region A and Region B. Voltages in Region A generate currents that relate to the TCA cycle, and Voltages in Region B are thought to prevent the TCA cycle from working.

Figure 3 A. The Bacteria were exposed to either 0.0V or 0.4V, and the current was measured over a series over hours. The CV points shown on this graph will be needed later, so I will be referring back to this graph. But it basically reinforces the fact that current flows better at 0.0V than at 0.4V. You may notcie that it takes about 10 hours for the current of the bacteria grown at 0.4V to decrease, suggesting that the TCA cycle can run on for a while in this more reducing environment.

Figure 3 B. This graph is the same as the previous graph, but they add malonic acid partway through, which completely ablates the current flowing through bacteria grown at 0.V. A short peak is observed in the 0.4V, probably because the addition of a new compound may be enough to cause perturbation to the current.

Figure 3 C. The researchers then measured the current of their TCA inactivated mutants, and found there was no difference in the flow of current when exposed to 0V or 0.4V, again pointing to the current flow being dependant on the TCA cycle being active.

This reiterates the previous point, and demonstrates how quickly the current disappears after TCA inhibitors are added. You should also note that current still flows in bacteria grown at 0.4V, and the reasons for that should become clear in the next figure.

Now we need to ask how these electrical changes relate to changes within the actual bacteria. So they measure the mRNA levels of specific genes for proteins involved in bacterial respiration. This allows researchers to measure what proteins bacteria are making in real time, and thus observe how the bacterial metabolism is reacting to the changes in voltage.

Figure 4 A. Here they measured the levels of Succinate dehydrogenase (sdh) mRNA, which encodes a protein that plays a key role in the TCA cycle. we can see that bacteria grown at 0V are making more sdh than bacteria grown at 0.4V, in keeping with the idea that TCA cycle is downregulated at the higher voltage.

Figure 4 B. They measured the levels of isocitrate dehydrogense mRNA, which encodes a protein that is important for a different part of the TCA cycle. We see the same effect here.

Figure 4 C. Here, they measured Lactate dehydrogenase mRNA, which encodes a protein that is not involved in the TCA cycle, and instead is involved in fermentation. This reaction allows for respiration even when oxygen isn't present as an electron acceptor. In this case, we see that Shewanella has adapted to life at the higher voltages by upregulating fermentation, allowing anaerobic respiration to occur. This explains the current difference at the end of the graph in Figure 3C. Bacteria grown at 0.4V adapt by stopping the TCA cycle, and increasing fermentation, and thus still produce a flow of electrons, whereas bacteria gown at 0V suddenly exposed to malonic acid have the whole TCA cycle abruptly shut down without fermentation there to pick up the slack.

Cyclic Voltammetry

Cyclic voltammetry is a method through which people can research reversible reduction/oxidation processes in chemicals. It involves slowly increasing the voltage until a certain threshold (in this case 0.4-0.5V) and then reversing it in the opposite direction. The initial positive peak is known as the "Peak Anodic Current" and the negative peak is known as the "Peak Cathodic Current" because effectively the electrode has changed from positive to negative.

In reactions that are completely reversible, you see a a symmetrical wave pattern, in which the trough and the peak approximate similar levels. If there is some irreversible component, or some other issue , it can cause asymmetric plots.

The values you get are somewhat dependant on the time it takes for the system to go through a cycle (known as the experiments' Scan Rate (mV/s)). If you use a high scan rate, it can cause an increase in the measured current. The standard scan rate used for bacteria tends to be 1-10mV/s, which is slower compared to looked at samples of pure chemicals. This is because respiration is a multi-step reaction, and you want to have at least on sequence of reactions to occur at a specific voltage. If the voltage changes at a rate that is faster than the time it takes for respiration to occur, then one part of the reaction will be occurring under different redox conditions than the other, and thus would be kind of messed up.

The researchers used cyclic voltammetry to measure the peak anodic current of bacteria sampled from those points on Figure 3A, CV-1, CV-2 and CV-3. CV-1 samples are from bacteria grown at 0V in the later stages of an experiment. CV-2 are bacteria grown at 0.4V at a very early time point of an experiment, and CV-3 are from bacteria grown at 0.4V at a late time period of an experiment

Figure 5 A.They found that the traces for CV-1 and CV-2 were identical, but the peak for CV-3 was larger than either of them. The bacteria that had adapted to the 0.4V were measured to have a much higher peak anodic current than the others.

Figure 5 B They repeated the experiment they did in Figure 3, but they added the malonic acid much earlier to show that the TCA cycle is still active for at least two hours after the 0.4V potential is applied to it.

Figure 5 C. This time they applied Cyclic Voltammetry to mutant without a TCA cycle cultured at 0.4V and 0.0V. They found that the Peak Anodic Current obtained from these bacteria showed a similar pattern to the bacteria with an intact TCA cycle. This suggests that the cyclic Voltammetry readings are not affected by TCA cycle, but can measure the degree to which bacteria adapt to the higher voltages.

Comments

The main reason why I don't think the Cyclic Voltammetry could tell the researchers anything about the TCA cycle was that they used a scan rate of 50mV/s as a scan rate.

You may recall that the standard scan rate for whole cell bacteria tends to be 1-10mV/s, because whole cells use multiple step reactions in order to respire. Changing the voltage between periods where it can work to periods when it cannot work in a time to short for a single turn of the cycle to occur effectively inhibits it. It's likely that the changes they do see are because the cells have adapted their metabolism to undergo the much quicker process of fermentation.

The 50mV would be okay if there was some justification, if they had tried this out at lower scan rates and slowly ramped them up, and found there was no difference in the trace they obtain. But there is no such justification in the paper. In fact, I was hard pressed to find justification for the scan rates in any of the other papers I could get my hands on on this subject. What if even a slow rate of 1mV/s is too quick for the TCA cycle to be measured ? .

Aside from that, there really is not much actual microbiology in this paper. The fact that we are dealing with bacteria is almost incidental to the way in which they are used here. we don't really get much idea of their growth phase, or the whether they form biofilms on these electrodes, or whether those biofilms change in response to the changing voltages. Why do the bacteria appear to be more reactive to voltage changes at 20 hours as opposed to 5 hours ? Is it because there are more there , or because they have changed and adapted in some way ?

Summary

This paper makes the case that the TCA cycle is regulated by the oxidative environment around it, and demonstrates it.

I just don't think they have sufficiently explained or justified Figure 5 enough for me to trust it, not to mention that an examination of the background literature suggest that there is a serious methodological flaw in it.

Thus, you can tell what I think of any conclusions about the TCA cycle they draw from that particular data.